Multiphoton excitation or absorption is the near simultaneous absorption of two or more photons to reach a reactive, excited state that is energetically inaccessible by the absorption of a single photon of the same energy. Multiphoton excitation, an important depth-sectioning tool in fluorescence microscopy,1,2 relies on the large power density present at the focal point of a pulsed laser to excite chromophores using two or more low-energy photons. The process can be thought of as the near-simultaneous absorption of two or more photons by a single molecule, a phenomenon that is only possible under conditions of very high laser power and tight beam focusing. For this reason, multiphoton excitation occurs only in a small volume around the focal point of the irradiating laser, while chromophores elsewhere in the laser path are exposed only to isolated low-energy photons. The three-dimensional control of excitation provided by this technique has made it very useful in micro-lithography, in which objects (including photonic devices) with very small feature sizes can be made by the photo-initiated cross-linking of oligomers in resin form.3 Recently, the formation of solid structures by multiphoton-induced cross-linking of soluble proteins in aqueous media has also been described.4,5 
The photochemical patterning of agarose hydrogels covalently modified with S-2-nitrobenzylcysteine has been previously reported6. The photochemical patterning of hyaluronan hydrogels covalently modified with S-2-nitrobenzylcysteine has also been described in a publication co-authored by one of the present inventors7 (the subject matter of these publications is incorporated by reference into the present application). Irradiation of these hydrogels with a conventional ultraviolet (UV) laser removed 2-nitrobenzyl protecting groups along the path of the laser beam, providing cylindrical volumes of free thiols through the full thickness of the hydrogel samples. Treating the patterned gels with maleimide-modified oligopeptides then resulted in the covalent immobilization of these molecules in the irradiated volumes via Michael-type addition, providing bulk substrates with cell-adhesive, cylindrical volumes defined in a purely chemical fashion (i.e., without altering the local mechanical properties).
6-bromo-7-hydroxycoumarin (Bhc) chromophore8 is well-attested as an efficient, multiphoton-labile (δu˜1 GM at 740 nm) protecting group for amines8, alcohols and phenols9, aldehydes10, and diols,11,12 and has also been used extensively as a phototrigger inside biological systems.8,13 